1. Field of the Invention
The present invention relates to phospholipase A1 mutants and a process for preparing the same, more specifically, to thermostable phospholipase TA3 and TA13 which are mutants of phospholipase A1 catalyzing the reaction of hydrolysis/synthesis of phospholipids, genes encoding the same, microorganisms transformed with recombinant expression vectors comprising the mutant genes and a process for preparing phospholipase A1 mutants therefrom.
2. Background of the Invention
It has been known that: lysophospholipid not only plays a role in platelet aggregation, but also mediates physiological activity such as signal transduction in animal (see: Durieux and Lynch, Trends Pharmacol. Sci., 14: 249, 1993), and also functions as a plant hormone to prevent plants or fruits from over-ripening (see: U.S. Pat. No. 5,126,155). Since lysophospholipid is highly soluble in water and forms stable emulsion under various hydrogen ion concentrations and broad range of temperature and has stability in the presence of magnesium or calcium ion, it has been applied to many industrial uses as an emulsifier such as pharmaceutics, cosmetics and food processing.
In biochemical pathway, lysophospholipid is formed via hydrolysis of phospholipid by phospholipase A1: that is, phospholipase A1 hydrolyzes 1-fatty acyl group (or fatty acyl group in the sn-1 position) of phospholipid to form lysophospholipid and fatty acid. The phospholipase A1 is an essential enzyme in the synthesis of phospholipids such as polyunsaturated fatty acids(PUFA) such as DHA or EPA. In physiological aspects, phospholipase A1 is related to human phospholipidosis caused by accumulation of phospholipid in lysosome due to the inhibition of phospholipase activity by cationic amphiphilic drug(CAD) (see: Reasor et al., Proc. Soc. Biol. Med., 212:297-304, 1996). Although phospholipase A1 has been isolated from a variety of sources such as mammals, snake toxins, bee toxins and microorganisms including Serratia sp. and Aspergillus sp., low stability of the enzymes hampered their application to biological processes.
The stability of enzyme is one of the most crucial factors in biological process where the enzyme is employed as a biological catalyst. Especially, the efficiency of biological catalysis performed at high temperature is shown to be relatively higher than that performed at low temperature. Hence, the thermostability of enzyme is the major concern in the biological process. In addition, enzyme reaction at high temperature has several advantages over enzyme reaction at room temperature or low temperature like high substrate solubility, reduced microbial contamination, lower viscosity of the reaction mixture, etc. Therefore, development of thermostable enzyme is necessary for conducting efficient biological process and for applying it for other related industry. Moreover, improved thermal stability of the thermostable enzymes is known to confer better structural stability at room temperature and confer higher resistance to denaturing factors such as organic solvents, extreme hydrogen ion concentrations and protein denaturants.
In view of the foregoings, in the preparation of phospholypase A1 for producing lysophospholipid, it is very important to prepare enzymes with improved thermal stability to enhance their enzyme activity. However, the methods proposed for synthesis of mutants of advantageous enzyme proteins, e.g., modification of protein tertiary structure by introducing disulfide bond, cross-link, salt bridge or metal binding site, are inappropriate for construction of mutants of highly active phospholipase A1 described above. Since the said modification methods can be applied only after the tertiary structure of enzyme is identified, it is impossible to apply the above methods to phospholipase A1 whose tertiary structure is not clearly understood.
Under these circumstances, it is urgently required to improve thermostability of phospholipase A1 for its universal use in biological processes. Therefore, there are strong reasons for exploring and developing novel phospholipase A1 mutants which have improved thermostability and enhanced catalytic activity as well.
The present inventors have made an effort to prepare phospholipase A1 mutants with improved thermostability which can be applied to various biological processes, and prepared phospholipase A1 mutants with substantially improved thermostability compared with the naturally occurring enzyme by introducing random mutations into the phospholipase A1 gene using a recombinant vector containing naturally occurring phospholipase A1 gene derived from Serratia sp. as a template for mutagenic polymerase chain reaction (PCR), followed by transforming the recombinant expression vector into E. coli. 
The first object of the invention is, therefore, to provide phospholipase A1 mutants derived from Serratia sp. strain MK1.
The second object of the invention is to provide genes encoding the mutants.
The third object of the invention is to provide recombinant expression vectors comprising the mutated genes.
The fourth object of the invention is to provide E. coli strains transformed with the recombinant expression vectors.
The fifth object of the invention is to provide a process for preparing phospholipase A1 mutants.